Over the past several years, we have been studying two phenomena in cloned populations of CD4 positive T lymphocytes referred to as costimulation and anergy. Both affect the production of the T cell growth factor interleukin-2 (IL-2) produced by these cells. Costimulation entails a 30 to 100-fold enhancement of IL- 2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen-specific receptor. In this case, subsequent stimulation of IL-2 production is inhibited 10-50-fold. Our goals are to try and understand the molecular mechanisms behind these two phenomena and to explore their relevance in vivo. Recently, we have set up a new model for studying tolerance to persistent low dose antigen in vivo, which results in the generation of a large number of anergic T cells. We inject CD4+, cytochrome c-specific T cells from a T cell receptor transgenic mouse on a Rag2-/- background (a monospecific T cell population) into a second transgenic mouse (RO) expressing the cytochrome c antigen under the control of the MHC class I promoter and an immunoglobin heavy chain enhancer. Within 24 hours after transfer, the T cells are all activated by the antigen (as evidenced by an increase in size and expression of CD69), and proliferate extensively for several days, increasing in number about 100-fold. This expansion is followed by a deletional phase during which 50% of the cells disappear. Finally, the population reaches a steady state level in which the cells appear to be refractory to restimulation in vivo and in vitro. In this adaptive tolerant state, cytokine responses to high doses of antigen in vitro are inhibited 90%. Expression on restimulation of early activation markers, such as CD69 and CD25, is also greatly impaired, and biochemical studies of signal transduction show an inhibition of Zap70 and LAT phosphorylation following T cell receptor engagement. This curtails the activation of phospholipase c gamma and prevents mobilization of intracellular calcium and NF-AT to the nucleus. This hyporesponsive state is reversible if the cells are transferred again into a second host not expressing the antigen. Interestingly, if the retransfer is into a host expressing the antigen, the cells are blunted in their in vivo proliferation and slowly decrease their IL-2 and IFN gamma production by another 6-10 fold over 3-4 weeks. This deeper state of anergy suggested that the tolerance process is adaptable to different levels. To test this idea more directly we studied a second transgenic mouse (SPK) expressing 3 fold lower amounts of antigen. TCR transgenic CD4 T cells injected into this SPK mouse also proliferated and entered into an anergic state; however, when the cells were subsequently retransferred into the first antigen transgenic (RO) mouse the T cells proliferated better than if they had first been tolerized in the RO host and then retransferred into it as mentioned above. This experiment shows for the first time that peripheral T cells can adapt their thresholds of activation to different levels depending on the concentration of antigen that persist in their environment. Preliminary biochemical studies suggest that subtle modifications in the ability to activate the MAP kinase pathway may account for these differences.